In article <2qbv22$jnr at nntp.ucs.ubc.ca>, Sarven Sabunciyan
<sarven at ecc.ubc.ca> wrote:
> Dear netters,
>> After several years of doing Northerns we have decided to switch to
> the RNAse protection assay. I was just wondering about what the in
> vitro transcription procedures or kits people used to make the RNA
> probe. I was also wondering how people isolate their RNA probe. I
> am especially curious about if the thickness of the polyacrylamide
> gel causes a problem with the resolution necessary or the eluting of
> the probe. Any sort of helpful hint or favorite procedure would be
> greatly appreciated.
>> Thanks a lot
>> Sarven
We use the Promega Riboprobe System II which is pretty good. We've
replaced the polymerases with Boehringer equivalents. One point, if your
P32 CTP is old ( ie over about a week) you may get inhibition of RNA
synthesis by the break down products. Also, if your probe is long you may
find it gets broken up if you keep it, my probe is about 150nt and I don't
have this problem.
We purify the probe by DNaseI treatment (to remove template), followed by
passage through two microselect spin columns (from 5 prime-3 prime) and a
phenol chloroform. We then run a bit out to check the purity, and
scintillation count!! DON"T USE THE AMBION RPA II KIT, ITS CRAP!!
For the protection assay itself I would advise against using RNase1, we did
and its rubbish!!! We use a mixture of RNaseA and RNase T1, which I
haven't had any problems with.
If you need any more info drop me a line and I'll send you a copy of my
"Donkey Sheet" with the exact method on it!!!
Bye.....Kate